Name: Mouse Bladder Wall Injection (Video) | JoVE Journal
Uploaded: 2011-07-12T12:00:00.000+00:00
Duration: 3 min 48 s
Description: Learn a microsurgical technique for precise cell injection into the mouse bladder wall. This method enables targeted implantation to study bladder stem cells and cancer biology with improved localization.

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We gratefully acknowledge the support of a Pilot grant from the Stanford Pediatric Research Fund and K08DK087895-01 from the NIDDK.

1. Mouse Bladder Wall Injection
Choice of mouse strain, age, and sex is dictated by experimental needs. We use mice between 8 and 12 weeks of age, since this is a window of immunological maturity prior to senescence. As a general guideline, mice should arrive at least one week prior to experimental manipulation in order to avoid stress-induced confounding factors.
<ol>
<li>Clean the surgical table surface with soap and water.</li>
<li>Wipe the surgical table surface with Cide Swipes or antiseptic wipes.</li>
<li>Autoclave clean surgical instruments prior to use in surgery.</li>
<li>In addition, sterilize surgical instruments with hot bead sterilizer immediately prior to use, as well as between animals during surgery.</li>
<li>Clean 100 μL Hamilton syringes and 29 or 30 gauge (1/2 inch long) needles by repeated aspiration and injection with absolute alcohol before first use and at the end of the final surgical procedure.</li>
<li>Wash and rinse Hamilton syringes with sterilized phosphate-buffered saline between each animal.</li>
<li>Anesthetize mice by placing them in an isoflurane induction chamber, with the isoflurane set between 2-5%.</li> 
<li>Once general anesthesia is achieved, remove the mouse from the chamber and place in the supine position with a warm pad underneath to help maintain normal body temperature.</li>
<li>Achieve maintenance anesthesia by placing the animal’s snout in a nozzle containing vaporize isoflurane (titrated from 1-3% as necessary to maintain appropriate anesthesia).</li>
<li>Shave the abdominal skin with clippers.</li>
<li>Use a disposable, sterile surgical drape to cover the anus to prevent fecal contamination during surgery.</li>
<li>Use a second disposable, sterile surgical drape to cover the surgical field (lower abdomen).</li>
<li>Prep the abdomen with three pieces of Betadine-soaked gauze. Repeat this step three times.</li>
<li>Using a dissecting microscope for magnification, make a lower midline abdominal incision with scissors.</li>
<li>Expose the bladder.</li>
<li>If the bladder is full, partially decompress it by gentle downward pressure at the dome.</li>
<li>Inject the sample solution (up to 50 μL), using a 29 or 30 gauge needle and syringe, into the wall of the bladder dome (intramural injection) with the bevel of the needle facing upwards.</li>
<li>Push the plunger of the syringe to inject the sample solution into the bladder wall. A well-localized bleb is an indication of successful injection of the cells into the bladder wall.</li>
<li>Remove the syringe, close the incision with wound clips and allow the mouse to recover on a warming pad.</li>
</ol>
2. Representative Results: 
A well-localized bleb that does not leak fluid and stays stable in size is an indication of successful injection of the cells into the bladder wall (Figure 1). Histological analysis can be performed to confirm the presence of the injected cells in the bladder wall.
<img src="/files/ftp_upload/2523/2523fig1.jpg" alt="Animal dissection; syringe application; anatomical study; research method; educational experiment." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Example of successful bladder wall injection using India ink for illustration.
<img src="/files/ftp_upload/2523/2523fig2.jpg" alt="Mouse bladder wall injection process diagram; midline laparotomy, syringe, cell solution procedure." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Schematic of experimental procedures.

<ol><li>Dinney, C. P., Fishbeck, R., Singh, R. K., Eve, B., Pathak, S., Brown, N., Xie, B., Fan, D., Bucana, C., Fidler, I. J., Killion, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+Characterization+of+Metastatic+Variants+from+Human+Transitional+Cell+Carcinoma+Passaged+by+Orthotopic+Implantation+in+Athymic+Nude+Mice%5BTitle%5D)+AND+%22The+Journal+of+Urology%22%5BJournal%5D)">Isolation and Characterization of Metastatic Variants from Human Transitional Cell Carcinoma Passaged by Orthotopic Implantation in Athymic Nude Mice.</a> The Journal of Urology. 154, 1532-1538 (1995).</li>
<li>Singh, A. V., Franke, A. A., Blackburn, G. L., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Soy+Phytochemicals+Prevent+Orthotopic+Growth+and+Metastasis+of+Bladder+Cancer+in+Mice+by+Alterations+of+Cancer+Cell+Proliferation+and+Apoptosis+and+Tumor+Angiogenesis%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Soy Phytochemicals Prevent Orthotopic Growth and Metastasis of Bladder Cancer in Mice by Alterations of Cancer Cell Proliferation and Apoptosis and Tumor Angiogenesis.</a> Cancer Res. 66, 1851-1858 (2006).</li>
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<li>Miyake, H., Hara, I., Yamanaka, K., Gohji, K., Arakawa, S., Kamidono, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overexpression+of+Bcl-2+Enhances+Metastatic+Potential+of+Human+Bladder+Cancer+Cells%5BTitle%5D)+AND+%22British+Journal+of+Cancer%22%5BJournal%5D)">Overexpression of Bcl-2 Enhances Metastatic Potential of Human Bladder Cancer Cells.</a> British Journal of Cancer. 79, 1651-1656 (1999).</li>
<li>Chancellor, M. B., Yokoyama, T., Tirney, S., Mattes, C. E., Ozawa, H., Yoshimura, N., Groat, W. C. de, Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preliminary+Results+of+Myoblast+Injection+into+the+Urethra+and+Bladder+Wall%3A+a+Possible+method+for+the+Treatment+of+Stress+Urinary+Incontinence+and+Impaired+Detrusor+Contractility%5BTitle%5D)+AND+%22Neurourol+Urodyn%22%5BJournal%5D)">Preliminary Results of Myoblast Injection into the Urethra and Bladder Wall: a Possible method for the Treatment of Stress Urinary Incontinence and Impaired Detrusor Contractility.</a> Neurourol Urodyn. 19, 279-287 (2000).</li>
<li>Dinney, C. P., Tanguay, S., Bucana, C. D., Eve, B. Y., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravesical+Liposomal+Muramyl+Tripeptide+Phosphatidylethanolamine+Treatment+of+Human+Bladder+Carcinoma+Growing+in+Nude+Mice%5BTitle%5D)+AND+%22J+Interferon+Cytokine+Res%22%5BJournal%5D)">Intravesical Liposomal Muramyl Tripeptide Phosphatidylethanolamine Treatment of Human Bladder Carcinoma Growing in Nude Mice.</a> J Interferon Cytokine Res. 15, 585-592 (1995).</li>
<li>Mohamedali, K. A., Kedary, D., Sweeney, P., Kamaty, A., Davisy, D. W., Evey, B. Y., Huangy, S., Thorpez, P. E., Dinney, C. P., Rosenblum, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Vascular-targeting+Fusion+toxin+VEGF121%2FrGel+Inhibits+the+Growth+of+Orthotopic+Human+Bladder+Carcinoma+Tumors%5BTitle%5D)+AND+%22Neoplasia%22%5BJournal%5D)">The Vascular-targeting Fusion toxin VEGF121/rGel Inhibits the Growth of Orthotopic Human Bladder Carcinoma Tumors.</a> Neoplasia. 7, 912-920 (2005).</li>
<li>Slaton, J. W., Perrotte, P., Inoue, K., Dinney, C. P., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interferon-%CE%B1-mediated+Down-Regulation+of+Angiogenesis-related+Genes+and+Therapy+of+Bladder+Cancer+Are+Dependent+on+Optimization+of+Biological+Dose+and+Schedule%5BTitle%5D)+AND+%22Clinical+Cancer+Research%22%5BJournal%5D)">Interferon-α-mediated Down-Regulation of Angiogenesis-related Genes and Therapy of Bladder Cancer Are Dependent on Optimization of Biological Dose and Schedule.</a> Clinical Cancer Research. 5, 2726-2734 (1999).</li>
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<li>Chan, E., Patel, A., Heston, W., Larchian, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+Orthotopic+Models+for+Bladder+Cancer+Research%5BTitle%5D)+AND+%22BJU+International%22%5BJournal%5D)">Mouse Orthotopic Models for Bladder Cancer Research.</a> BJU International. 104, 1286-1291 (1988).</li>
<li>Russell, P. J., Jelbart, M., Wills, E., Singh, S., Wass, J., Witherspoon, J., Raghavan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+and+characterization+of+a+new+human+bladder+cancer+cell+line+showing+features+of+squamous+and+glandular+differentiation%5BTitle%5D)+AND+%22Int.+J.+Cancer%22%5BJournal%5D)">Establishment and characterization of a new human bladder cancer cell line showing features of squamous and glandular differentiation.</a> Int. J. Cancer. 41, 74-74 (1988).</li>
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No conflicts of interest declared.

Mouse bladder wall injection allows for the implantation of specific cells into defined areas of the bladder wall. This technique has wide applications for mouse models of bladder cancer, smooth muscle, and stem cell biology. By definition, mouse bladder wall injection facilitates introduction of bladder cancer or stem cells into an orthotopic location so that their growth and differentiation can be studied in a physiologically relevant anatomic context. In fact, Dinney et al. first described this technique in order to s...

<table><tbody><tr><td>Isoflurane (Aerrane)</td><td>Baxter Internationl Inc.</td><td>NDC 10019-773-60</td><td></td></tr><tr><td>29 or 30 gauge needles</td><td>Hamilton Co</td><td>7803-06 or 7803-07</td><td></td></tr><tr><td>100 microliter syringe</td><td>Hamilton Co</td><td>7656-01</td><td></td></tr></tbody></table>

mouse bladder wall injection

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

The overall goal of this procedure is to introduce cells into defined areas of the bladder wall. This is accomplished by first making an abdominal incision to expose the bladder. The next step of the procedure is to insert the bevel of the needle intramurally.The final step of the procedure is to push the plunger of the syringe to inject the cells of interest into the bladder wall. Successful implantation of cells is marked by a well localized bleb that does not leak fluid and stays stable in size. The main advantage of this technique over existing methods like transurethral inoculation of mice with cells, is that it enables one to direct where the cells will implant themselves within the bladder.Visual demonstration of li muscle is critical as the injection stay are hard to learn. Knowing the correct angle and the entry position of the needle into the bladder is essential to the technique success. Select the age, sex, and strain of mice based on needs of the experiment here.Eight to 12 week old mice are used to begin clean the surface of the surgical table with soap and water. Next, wipe the surgical table surface with sideswipes or antiseptic wipes. Surgical instruments should be sterilized in an autoclave prior to surgery.Wipe the instruments with 70%ethanol just prior to use and re-sterilized with a hot bead sterilizer between individual surgeries. Clean a 100 microliter Hamilton syringe attached to a 30 gauge needle by repeated aspiration of absolute alcohol, followed by sterilized phosphate buffered saline. Place an anesthetized animal in the supine position on a warm pad to help maintain normal body temperature.Maintain a sufficient level of anesthesia by placing the animal snout in a nozzle containing vaporized isof fluorine. Next, remove the hair from the abdominal skin with clippers. Use a disposable sterile surgical drape to cover the anus to prevent fecal contamination during surgery.Place a second surgical drape over the lower abdomen and expose a surgical window. Sterilize the surgical site with repeated applications of Betadine and alcohol using a dissecting microscope for magnification. Make a lower midline abdominal incision with scissors.Use gentle pressure on the abdomen to expose the bladder. Since the bladder is usually partially or completely full pressure on the abdomen will preferentially push away other organs and allow the bladder to emerge the incision. If the bladder is full, apply gentle downward pressure at the dome to partially decompress it.Fill the sterile Hamilton syringe with 50 microliters of the sample solution. Insert the tip of the needle with the bevel facing up into the wall of the bladder and inject the sample solution. A successful injection is marked by a well localized bleb that does not leak fluid and maintains a stable size.After the injection close the incision with wound clips. Allow the master to recover on a warming pad One master. This technique can be done in 10 to 15 minutes if it is performed properly.Success can be confirmed through histological analysis of the bladder at serial time points post injection.

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Learn a microsurgical technique for precise cell injection into the mouse bladder wall. This method enables targeted implantation to study bladder stem cells and cancer biology with improved localization.

Mouse Bladder Wall Injection

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